(a) Field of the Invention
The present invention relates to a method of peptide sequencing by MALDI (matrix-assisted laser desorption ionization) tandem mass spectrometry in which an ionization status and chemical property of peptide are modified by at least one chemical modification selected from the group consisting of guanidination and esterification.
The present invention also relates to a method for preparing a sample for MALDI mass spectrometry including coating charged particles formed by electrospraying a sample solution under atmospheric pressure on a MALDI substrate using an ion-optical focusing instrument and an apparatus for electrospraying and focusing for the same.
(b) Description of the Related Art
At present, the most frequently used technique for identifying and characterizing proteins is based on mass spectrometry. However, the current technique is limited in detection sensitivity, making it applicable only to large volume of protein samples or readily ionized samples. Further, the sequencing requires a long time and only a limited number of proteins can be analyzed. Thus, researches are underway to find techniques capable of analyzing a large volume of samples in a short time and of improving detection sensitivity.
Tandem mass spectrometry is used in peptide sequencing of peptides. A peptide to be sequenced is selectively fragmented and the mass of the resultant daughter ions is measured to obtain the amino acid sequence of the peptide. The tandem mass spectrometry data are utilized to identify the particular protein by comparison with a gene sequence database. However, it is known that the reliability of this process is only 10 to 35%.
Such low reliability results from cleavage at a particular amino acid residue of a peptide or wrong daughter ion labelling caused by internal fragmentation. According to a recent research, monovalent (+1) ions mainly generated by matrix-assisted laser desorption ionization (MALDI) experience less internal fragmentation during tandem mass spectrometry than multivalent (+n, n≧2) ions mainly generated by electrospray ionization (ESI).
However, monovalent ions show a large difference in detection sensitivity depending on the amino acid composition of the particular peptide and cleavage tends to be concentrated around a specific amino acid residue, giving non-uniform daughter ion distribution. Despite these problems, MALDI is better suited for protein research, in which only a small amount of a sample is available, than ESI because it requires only a small volume of a sample, is not significantly affected by salts, and is convenient in spectrum analysis. Thus, a lot of attempts have been made in order to solve the problem in sequencing monovalent peptide ions generated by MALDI.
MALDI-MS is a cutting-edge soft ionization technique used in mass spectrometry, allowing, among other things, analysis of biomolecules (including proteins) and polymers. In contrast with ESI-MS, MALDI-MS is less affected by contamination of the sample from buffer solution, salt, bleaching agent, etc., requires a smaller sample, enables quick analysis, and makes it possible to perform a variety of re-analysis, including peptide sequencing, until the sample is depleted. However, because MALDI-MS requires a solid sample, it is not readily compatible with separation techniques treating liquid samples, in comparison with ESI-MS. To overcome this problem, continuous-flow probes or aerosol interfaces have been used to couple LC (liquid chromatography) with MALDI-MS and enable on-line MALDI-MS measurement. However, they have not satisfied the requirements in resolution, accuracy, and detection sensitivity needed for proteomics research. Although on-line MALDI-MS using moving wheels or moving balls in a vacuum container showed some improvement in resolution and detection sensitivity, it was restricted to a flow rate of nanoliters per minute and had no practicability or applicability.
Researches have been made on off-line MALDI-MS, in which, differently from on-line MALDI-MS, effluent from LC or capillary LC is fractionally collected to prepare samples adequate for MALDI-MS. The early technique used for off-line MALDI-MS analysis was fractionally collecting effluent using micro-vials or a 96-well micro-titer plate, concentrating each fraction by, for example, SpeedVac, mixing them with a matrix, and loading on a MALDI plate. This process was problematic in that a long time is required and automation is difficult. In addition, loss of the sample during concentration and sample-matrix preparation was a fatal problem. To solve these problems, several groups have developed automation methods for loading LC effluent directly on a MALDI plate as a spot or a track using such techniques as micro-dispenser piezoelectric flow, blotting, vacuum-assisted deposition, electric field-driven droplet deposition, heated nebulization, heated droplet delivery. However, these methods are adequate only for nanoflow or very slow flow. Besides, as heat or an impact is directly applied to the sample, it may be modified physically or chemically. Also, loss of sample is inevitable. Particularly, all the techniques developed thus far provide too large spots, which is the typical problem of MALDI sample preparation, resulting in dearth of sample per unit area and poor in-spot homogeneity or spot-to-spot homogeneity. As a result, a lot of time has to be spent to find out the “sweet spot”, or the sample/matrix crystal. Therefore, samples with smaller quantity or lower abundance tend not to be detected.
Also, because the sample/matrix crystal is too large, a lot of matrix ions are generated during laser desorption ionization, resulting in noise during the sample detection. With the conventional methods, signals of about 800 Da or less are not detected in general, because they are screened by the matrix ions.